Detection and quantitation of human papillomavirus type 16, 18 and 52 DNA in the peripheral blood of cervical cancer patients

OBJECTIVE: To prospectively evaluate the feasibility of detecting human papillomavirus (HPV) type 16, 18 and 52 DNA in the peripheral blood of patients with cervical cancer using real-time polymerase chain reaction (PCR) and to determine its prognostic importance. METHODS: Blood and cervical swab specimens from 135 consecutive patients with 60 invasive cervical cancers, 10 microinvasions, 20 cervical intraepithelial neoplasias (CIN) III, 10 CIN II, 10 CIN I and 25 controls were collected and examined for HPV type 16, 18 and 52 DNA using real-time PCR to investigate the prevalence and viral load of HPV DNA at the time of diagnosis and during follow-up in patients with positive blood samples. RESULTS: Of the 60 patients with invasive cervical cancer, 27% had positive test results for HPV DNA in blood samples in contrast to 0% of patients with microinvasions, CIN III, CIN II, CIN I and normal controls. The DNA detection rates of viral subtypes in blood samples of cervical cancer patients were 5% for HPV-16, 16.7% for HPV-18, 8.3% for HPV-52, 1.7% for both HPV-16 and HPV-18 and 1.7% for both HPV-18 and HPV-52, while the detection rates in cervical swab specimens were 36.2% for HPV-16, 15.5% for HPV-18 and 17.2% for HPV-52. During follow-up, 8 of 10 cervical cancer patients with viral DNA detected in blood within 3 months after treatment had recurrence, and a high percentage (87.5%, 7/8) of this recurrence involved distant metastases. CONCLUSIONS: In this study, real-time PCR detected HPV-16, -18 or -52 DNA in the peripheral blood of more than one-fourth of invasive cervical cancer patients. The association between risk of cancer recurrence and the amount of viral DNA detected in blood among cervical cancer patients after treatment is intriguing and deserves further investigation.     

Detection of human papillomavirus DNA in human cervical lesions by tissue in situ nucleic acid hybridization

Cervical cancer is one of the most common female cancers in Taiwan. Certain types of human papillomavirus (HPV) are frequently detected in the epithelial precancerous and cancerous lesions of the cervix. By the use of tissue in situ hybridization, we investigated the relationship of various types of HPV (group I, HPV-6 & 11, group II, HPV-16 & 18, group III, HPV-31, 33 & 35) with cervical condyloma, carcinoma as well as precancerous lesions. Group I HPV DNAs were mainly found in cervical condylomatous lesions (2/2) of the cervix and cervical intraepithelial neoplasia I (CIN I) (2/4), but were only occasionally found in CIN II (1/4), CIN III (1/9) or non-keratinized squamous cell carcinoma (1/15). HPV DNAs of groups II and III were mainly detected in lesions of CIN III (5/9) and invasive squamous cell carcinoma (large cell, keratinized type: 4/7; large cell, non-keratinized type: 11/15). HPV DNA sequences were invariably detectable only in the cell nuclei of condyloma or dysplastic epithelium or invasive carcinoma. However, they could not only be detected in the upper layer dysplastic cells and koilocytes but also in the well and poorly differentiated cervical cancer cells. The distribution of HPV DNA positive cells in the carcinomas fell into four different patterns: (1) upper zone and non-invasive regions of the carcinoma (11/22, 50%), (2) basal zone and invasive regions (2/22, 9%), (3) randomly scattered (7/22, 32%), and (4) extensively distributed over the whole tumor lesions (2/22, 9%). Thus, our results are consistent with a strong correlation between the presence of HPV-16, 18, 31, 33 and 35 and malignant conversion of cervical epithelial cells.     

Human papillomavirus genotype as a prognostic factor in carcinoma of the uterine cervix

The clinical implications of specific human papillomavirus (HPV) types in invasive cervical carcinomas are only now beginning to be appreciated. The objective of this study was to determine the clinical implications and prognostic value of the HPV genotype in cervical carcinomas. In this study, we employed an HPV DNA chip to detect the type-specific sequence of HPV from cervical swabs taken from women with biopsy-proven neoplastic lesions of the cervix. We divided the patients into four groups: HPV-negative, HPV-16-related, HPV-18-related, and intermediate risk type-related. Associations with clinicopathologic data (stage, histologic type, lymph node status, parametrial invasion, lymphvascular space invasion, tumor size, vaginal involvement) and overall survival were assessed. HPV DNA was detected in 81.4% of the patients, and 19.0% harbored multiple HPV variants. HPV-16-related was the predominant type and was detected in 47.4% (46/97) of the patients. The HPV-16-related types were detected more frequently in patients with squamous cell carcinomas, whereas the HPV-18-related types were more prevalent in cases of adenocarcinomas and adenosquamous carcinomas (P < 0.05). Otherwise, no significant correlations were detected between the HPV genotype and any other clinicopathologic parameters. After a median follow-up of 30 months, the 5-year survival rate was lower in the HPV-18-related patients, but this difference was not found to be statistically significant, according to the results of the log-rank test. We conclude that neither the presence nor type of HPV DNA bears any prognostic significance in cases of cervical carcinoma.     

Human papillomavirus DNA in adenocarcinoma in situ, microinvasive adenocarcinoma of the uterine cervix, and coexisting cervical squamous intraepithelial neoplasia

Previously, human papillomavirus (HPV) DNA, mainly HPV-18 DNA, was detected in more than 40% (17/40 cases) of invasive adenocarcinoma of the uterine cervix in our laboratory. In order to identify HPV DNA in the precursor lesions of adenocarcinoma of the cervix, 11 cases of adenocarcinoma in situ containing microinvasive adenocarcinoma and 10 cases of adenocarcinoma in situ were studied for the presence of HPV DNA by in situ hybridization using highly sensitive 3H-labeled HPV-16 and HPV-18 DNA probes. HPV types present in cervical squamous intraepithelial neoplasia (CIN) coexisting with adenocarcinoma in situ and microinvasive adenocarcinoma were also studied. Apart from the coexisting CIN II-III with glandular neoplasms, 48 cases of CIN III (severe dysplasia and squamous carcinoma in situ) removed by conization or hysterectomy and known to be free of adenocarcinoma were used for comparison. HPV DNA was detected in 64% of microinvasive adenocarcinoma, 70% of adenocarcinoma in situ, and 63% of the control CIN III. HPV-18 DNA was the preponderant type of HPV DNA found in adenocarcinoma in situ and microinvasive adenocarcinoma. All cases of HPV DNA-positive microinvasive adenocarcinoma contained the same type of HPV DNA as the lesions of coexisting adenocarcinoma in situ. CIN coexisting with microinvasive adenocarcinoma or adenocarcinoma in situ contained the same type of HPV as identified in the glandular lesions, whereas all of the HPV DNA-positive control CIN III cases contained HPV-16 DNA. These results suggest that adenocarcinoma in situ is a precursor lesion of adenocarcinoma of the cervix that contains HPV DNA, and that CIN coexisting with adenocarcinoma may be a result of a metaplastic process of adenocarcinoma or of bidirectional differentiation of the affected reserve cells.     

Mutational and functional analysis of HPV-16 URR derived from Korean cervical neoplasia.

OBJECTIVE: The YY1 mutation has been suggested as one of the indicators that explains development of cervical neoplasia by episomal-type HPV. To extend this hypothesis, we examined whether a mutation(s) in the YY1 site is functionally related to the invasiveness of cervical neoplasia and the physical status of HPV DNA. METHODS: The URR sequences were obtained by PCR amplification of HPV-16 genome from CIN and invasive cancer patients and cloned into pUC18 for sequencing and into pBLCAT8+ for functional CAT assay. RESULTS: Our previous data classified HPV-infected patients into three groups: 3 cancer cases carrying episomal HPV DNA; 12 cancer cases carrying integrated HPV DNA; 12 CIN cases carrying episomal HPV DNA. The specific variants in HPV-16 URR were found in Korean women: G-->A transition at nt 7520 (100%, 27/27), A-->C transition at nt 7729 (70%; 19/27), and G-->A transition at nt 7841 (78%; 21/27). Selective mutations were observed at the YY1 binding sites of HPV-16 URR in the 3 patients with invasive cervical cancer who have the episomal forms of HPV-16 DNA: A-->C transition at nt 7484 and G-->A transition at nt 7488 (YY1-binding site 2; from 7481 to 7489). Additionally, C-->T transition at nt 7785 (YY1-binding site 3; from 7781 to 7790) was found in 2 of 3 patients. No YY1 site mutations were detected in the 12 CIN patients and in the HPV-integrated invasive cancer patients. To determine whether these mutations have effects on the expression of HPV E6/E7 genes driven by URR, the transient transfection assay was employed using URR-CAT reporter plasmid. The relative activities of three URR mutants from episomal HPV-16 DNA of cervical cancers were two- to fourfold higher than that of the HPV-16 URR prototype. In contrast, the URRs from integrated HPV-16 DNA in cervical cancer and from episomal HPV-16 DNA in CIN, where no mutation of the YY1 binding site was detected, showed similar levels of promoter activity to that of the URR prototype. CONCLUSIONS: Our results support the hypothesis that the mutation at the YY1 binding site is functionally related to the development of cervical neoplasia caused by episomal HPV-16 DNA in Korean cervical cancer patients. Thus, mutation in the YY1 site of episomal HPV-16 URR may play a corresponding role of HPV integration in the progression of cervical cancer.     

Virological studies on generation of cervical carcinomas

1. Detection of HPV in cytologically normal cervices: We have detected by filter in situ hybridization human papillomavirus (HPV) types 6/11, 16 and 18 DNA sequences in 6(0.9%), 12(1.8%) and 4(0.6%), respectively, out of 666 swab specimens from normal cervices (mean age; 49.1 years). The positive rate for HPV 16 and HPV 18 was significantly lower compared with 27.1%(26/96) of CIN and 48.4%(15/31) of cervical cancers. HPV DNA occurred more often in women under 50 years old than in those 50 years old or older (5.1% versus 1.0%). 2. Detection of HPV in cervical intraepithelial neoplasia (CIN): Eighty nine patients with CIN I-III were examined by Southern blot analysis with HPV 11, HPV 16 and HPV 18 DNAs in stringent conditions (Tm -18 degrees C) and with HPV 16/18 mixed probe in relaxed conditions (Tm -37 degrees C). We found HPV 11, HPV 16, HPV18 and other types of HPV in 0%(0/37)/2.7%(1/37)/2.7% (1/37)/16.2%(6/37) of CIN I, 0% (0/11)/9.1%(1/11)/0%(0/11)/45.5%(5/11) of CIN II and 0% (0/41)/26.8%(11/41)/2.4%(1/41)/24.4%(10/41) of CIN III. 3. Detection of HPV in cervical carcinomas: One hundred sixty seven cervical carcinomas and 6 metastatic tumors from cervical carcinomas were examined by Southern blot analysis with HPV 16 and HPV 18 DNAs in stringent conditions (Tm -18 degrees C). HPV 16 and HPV 18 were found in 35.3% (59/167) and 7.2%(12/167), respectively. The incidence of HPV 16/18 was higher in the patients under 60 years old (55.1% [27/49]). HPV 18 was detected more often in adenosquamous carcinomas and adenocarcinomas (31.8% [7/22]) than in squamous cell carcinomas (3.4% [5/145]). Five out of 6 metastatic tumors were positive for HPV 16 or HPV 18. 4. Physical state of HPV DNA in CIN and cervical carcinomas: The physical state of HPV 16 and HPV 18 DNAs was determined by electrophoresis after digestion with uncut and single-cut enzymes and two-dimensional electrophoresis after digestion with uncut enzyme. Three out of 3 CIN had only free episomal HPV DNA. HPV 16 DNA was existed as free episomal DNA in 4, as free episomal DNA and integrated DNA in 7, as integrated DNA in 8 out of 19 cervical carcinomas. HPV 18 DNA was integrated into the host cell genome in 3 out of 3 cervical carcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)     

Presence of Oncogenic HPV DNAs in Cervical Carcinoma Tissues and Pelvic Lymph Nodes Associating with Proliferating Cell Nuclear Antigen Expression

The presence of oncogenic HPV DNAs (HPV-16/18) in cervical carcinomas and their normal and metastatic pelvic lymph nodes and the expression patterns of proliferating cell nuclear antigen (PCNA) in cervical carcinomas were retrospectively studied to elucidate the possible roles of them in malignant transformation and progression of the disease. HPV-16/18 DNAs were detected by polymerase chain reaction using HPV E6 type-specific primers in 79 patients with cervical cancer: 31 patients who had pelvic lymph node metastasis (group I) and 48 patients without pelvic lymph node metastasis (group II) who were proven by pathologic examination of surgical specimens. HPV-16 or -18 DNAs were detectable in cervical carcinoma tissues in 60 patients from 79 cervical cancer patients (75.9%; HPV-16 was 67.1% and HPV-18 was 8.9%). HPV DNAs were amplified from metastatic pelvic lymph nodes in 13 patients of group I (42%) and from nonmetastatic lymph nodes in 7 group I patients (22.5%). Recurrence was identified in 9 group I patients (29.0%) in 3 years of follow-up. HPV DNAs were amplified from nonmetastatic lymph nodes in 11 group II patients (22.9%). Two group II patients, who had HPV-16 DNA by PCR in nonmetastatic nodes, were recurrent. PCNA was overexpressed in 66.7% of HPV-16- or -18-positive cervical cancers and 16.7% of HPV-16- or -18-negative cervical cancers. However, the expression levels of PCNA in cervical cancers were not influenced by the presence of oncogenic HPV DNA or pathologic metastasis in the pelvic lymph nodes. In conclusion, HPV DNA could be amplified from some metastatic and nonmetastatic pelvic lymph nodes and the detectability of oncogenic HPV DNA in pelvic lymph nodes may represent the poor outcome in the treatment of disease. The expression of PCNA protein which was associated with presence of oncogenic HPV DNAs in cervical cancers, suggesting activation of S phase of cell cycle, may contribute to the malignant progression by HPV-16 or -18.     

Evaluation of adenoassociated virus 2 and human papilloma virus 16 and 18 infection in cervical cancer biopsies

OBJECTIVE: Protective roles of adenoassociated virus (AAV) 2 in cervical tumorigenesis are controversial. In an effort to clarify this issue, we tested prevalence of AAV 2 and human papillomavirus (HPV) infection in cervical lesions and adjacent normal tissues. METHODS: Tissues of cervical intraepithelial neoplasm (CIN) I (20 patients), CIN II (24 patients), CIN III (25 patients), and invasive cancer (23 patients) were investigated by microdissection and PCR using HPV-16-, HVP-18-, and AAV-2-specific primers. RESULTS: AAV 2 was detected in 11 out of 20 CIN I (55%), 21 out of 24 CIN II (84.5%), 13 out of 25 CIN III (52%), and 12 out of 23 invasive cancer cases (52.2%). However, HPV 16 was detected in none out of 20 CIN I, 2 out of 24 CIN II (8.3%), 6 out of 25 CIN III (24%), and 6 out of 23 invasive cancer cases (26.1%). HPV 18 was detected in 1 case in CIN II (4.2%) and 2 cases in CIN III (8%). In 92 perilesional normal tissues, AAV 2 was detected in 53 cases (57.6%), displaying 25% of CIN I, 83.3% of CIN II, 52% of CIN III, and 65.2% of invasive cancer. CONCLUSION:The differences in AAV 2 prevalence are not significant between CIN and normal tissues. However, differences in HPV 16 are significant in CIN III and invasive cancer, as compared to CIN I, CIN II, and normal, suggesting no significant correlation between AAV 2 and cervical cancer. Thus, these results support the notion that AAV 2 is not associated with cervical tumorigenesis.     

Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-ras oncogenes in invasive cervical carcinomas.

Invasive carcinomas of the uterine cervix of 38 patients were examined for the presence of human papillomavirus (HPV) genomes and for the state of the c-myc and Ha-ras oncogenes. A combination of Southern blot hybridization and polymerase chain reaction revealed the presence of the genome of HPV type 16 in 17 tumors (45%), that of HPV type 18 in 3 tumors (8%), and that of unknown types in 16 others (42%), while no viral DNA sequences were detected in 2 tumors. Of the 38 tumors, c-myc amplification was found in only 1 tumor, while there was no Ha-ras amplification. Overexpression of the c-myc gene was observed in 15 (44%) of the 34 tumors analyzed, while there was no overexpression of Ha-ras. Of the 23 squamous cell carcinomas analyzed, relapse-free rates at 24 months were 55% in tumors with c-myc overexpression and 100% in case of tumors with no c-myc overexpression, respectively. The results suggest the possibility that activation of the c-myc oncogene is involved in tumor progression.     

Prevalence of human papillomavirus types 16 and 18 in cervical adenocarcinoma and its precursors in Scottish patients

Our aim was to determine the prevalence of human papillomavirus (HPV) types 16 and 18 in cervical adenocarcinoma (and its precursors) in Scottish patients. Nucleic acid was extracted from paraffin-embedded, formalin-fixed tissues. We examined 119 cases of invasive adenocarcinoma, 20 cases of adenocarcinoma in situ, and 16 cases of normal glandular epithelium. HPV DNA was detected by polymerase chain reaction using type-specific primers for the E6 and E7 genes of HPV-16 and HPV-18 with conformation of HPV genotype by subsequent restriction fragment length polymorphism. HPV DNA was identified in 87 (62.6%) cases, with HPV-16 being detectable in 65 (47%) cases and HPV-18 in 41 (29%) cases. All the cases of normal tissue tested negative for HPV-16 and/or HPV-18. No significant relation between infecting HPV type (16 or 18) and subtypes of disease (within the invasive category and between the preinvasive and the invasive categories) was noted. Our findings support that HPV-16, along with HPV-18, are likely to play a significant role in the pathogenesis of cervical adenocarcinomas and that cervical cancer screening strategies that incorporate oncogenic HPV testing, and prophylactic vaccines that target these types, will be beneficial for the reduction of adenocarcinoma and associated glandular precursors.     

Detection of human papillomavirus types 16, 18 and 33 in exfoliated cervical cells by polymerase chain reaction]

Human papillomavirus (HPV) types 16, 18 and 33 were identified by means of the polymerase chain reaction using exfoliated cells from the uterine cervix in 361 patients. Of 261 patients without cervical lesions, 10(3.8%) patients had HPV DNA whereas 7(70.0%) of 10 patients with invasive cervical carcinomas had HPV DNA. The younger patients' group (29 year-old or less) without cervical lesions had a 6.5% HPV positive rate which was distinctly higher than the older patients' groups. No menopausal patient without cervical lesions had HPV DNA. In the cervical dysplasia group, the HPV DNA positive rate tended to be higher in the older patients. Type 16 was detected more often than types 18 or 33. However, the detectable incidence of type 16 in the follow up group was lower than in the cervical carcinoma groups. The younger patients without cervical lesions had a higher incidence of type 16 than the older patients. The younger patients with cervical neoplastic lesions had a lower incidence of type 16 than the older patients. These results suggest that type 16 has a higher frequency of cervical HPV infections than types 18 and 33. In addition, human papillomavirus is not the only causative factor in cervical carcinomas.     

Herpes simplex virus and human papillomavirus infection in cervical disease in Argentine women.

The aim of the present study was to determine that prevalence of herpes simplex virus (HSV) type 1 and 2 in cervical samples from Argentine women and to assess the role of HSV-2 in cervical cancer. A sample of 79 normal and 200 neoplastic cervical tissues (35 invasive cervical carcinomas, 75 high-grade squamous intraepithelial lesions, 79 low-grade squamous intraepithelial lesions and 11 abnormal squamous cells of undermined significance) was analyzed for herpes simplex and human papillomavirus DNA using the polymerase chain reaction method. Viral genotyping was performed by single strand conformation polymorphisms and restriction fragment length polymorphisms. The overall prevalence of HSV was 21.5% in controls and 29% in cases. Among women with normal cytology, herpes simplex prevalence in HPV positive (20.8%) women was approximately the same as in negative (21.8%) women. HPV- and age- adjusted ORs of high-grade squamous intraepithelial lesions and invasive cervical carcinomas for HSV-2 were 1.4 (p = 0.6) and 1.6 (p = 0.5), respectively. The obtained results indicated that herpes simplex virus may not be involved in cervical cancer development. Future investigations are needed to provided conclusive evidence on the role of this pathogen in cervical cancer.     

Detection of HPV DNA in the uterine cervical lesions by polymerase chain reaction and in situ hybridization]

Human papillomavirus (HPV) is found in close association with carcinogenesis of the uterine cervix. We applied a new in vitro gene amplification technology, the polymerase chain reaction (PCR) to detect HPV 16 and 18 in cervical exfoliated cells. HPV infections were detected in 5 (16%) of 31 women with no pathological lesions of the uterine cervix (normal), 16 (24%) of 67 with cervical intraepithelial neoplasia (CIN) and 6 (38%) of 16 with invasive cervical cancer. Moreover, 10% formalin-fixed and paraffin-embedded tissue sections were prepared from the uterine cervix of these 27 women with PCR-proven HPV infection and were examined for the histological localization of HPV-DNA by in situ hybridization with biotin-labeled DNA probes of HPV types 6/11, 16/18 and 31/33/35. HPV-DNA type 16/18 was detected in 3 of 5 normal women, 2 of 4 CINs I, 2 of 3 CINs II, 6 of 9 CINs III and 6 of 6 invasive cervical cancers. HPV-DNA type 6/11 was detected in 6 of 6 condylomas. Viral DNA sequence was detected in the superficial cells of CIN I and II, and it was distributed through entire thickness layer of undifferentiated cells derived from CIN III and squamous cell carcinoma. In addition, the staining intensity became weak as the lesion progressed. These differences between lesions might be due to the difference in the viral form in the nuclei, ie whether an episomal or integrated form. Thus, an in situ hybridization technique with a biotin-labeled DNA probe as well as the PCR method is useful for the detection of HPV in clinical samples.     

In situ hybridization analysis of HPV DNA in cervical precancer and cervical cancers from China

Summary A series of 103 cervical biopsies derived from 103 women during July 1958 to September 1963 from Beijing, China were investigated with in situ hybridization for the presence of HPV6, 11, 16, 18, 31 and 33 DNA. The mean age of the patients was 46.1 + 10.6 years with a range of 24–74 years. Morphological features of HPV infection were found in 80 (77.7%) biopsies. Invasive cervical cancer was diagnosed in 43 biopsies and cervical intraepithelial neoplasia CIN I, CIN II and CIN III in 9, 9, and 27 cases, respectively. A total of 63.1% (65/103) of the lesions had morphological features of HPV infections associated with CIN or invasive carcinomas. Altogether, 31.1% (32/103) of the biopsies were shown to contain HPV DNA. Of the cases showing HPV morphology, 43.1% were HPV DNA positive. HPV16 (30/32) was the most frequent type, followed by HPV11 and 18, whereas no lesions with HPV6, 31 or 33 were found. A total of 19/43 (44.2%) of the invasive carcinomas contained HPV DNA. HPV DNA positivity and the grade of CIN showed a statistically significant correlation (P=0.0011). Our study demonstrated the presence of HPV in cervical lesions among Chinese women in the late 1950's and early 1960's when a single sexual partner was the rule and also supports the concept that HPV has as an important etiological role in cervical cancer, the highest risk being associated with HPV type 16. The applicability of in situ hybridization in retrospective assessment is emphasized.     

No metastatic cervical adenocarcinomas in a series of p16INK4a-positive mucinous or endometrioid advanced ovarian carcinomas: an analysis of the AGO Ovarian Cancer Study Group.

Epithelial ovarian cancers may represent secondary manifestations of occult extraovarian carcinomas. Such tumors may be misdiagnosed as primary ovarian carcinomas clinically and pathologically. In a recent study, several cases of cervical cancer metastases with primary manifestation as mucinous or endometrioid ovarian carcinomas were described. In all cases, human papillomavirus (HPV) DNA and diffuse p16 immunostaining were detected in the ovarian tumor tissues. To estimate the frequency of occult metastatic endocervical adenocarcinomas in a series of mucinous or endometrioid ovarian carcinomas, 24 ovarian cancers with mucinous and 50 with endometrioid differentiation from the Arbeitsgemeinschaft Gynaekologische Onkologie OVAR-3 database were analyzed for HPV and p16 positivity. The p16 immunostaining was performed using the p16 histology kit (Dako, Glostrup, Denmark), and both nuclear and cytoplasmic staining results were considered positive. Human papillomavirus polymerase chain reaction analysis was performed using 2 consensus primer systems (GP5/GP6 and PGMY09/11) and HPV-16- and HPV-18-specific primers from the L1 and the E6 regions. Six (8%) of 74 tumors were p16-negative, 13 (18%) of 74 showed single positive cells, 28 (38%) of 74 showed focal homogeneous staining, and 27 (36%) of 74 showed complete diffuse staining. In several independent amplifications of different regions of the HPV genome, none of the 73 tumors available for analysis showed the presence of HPV DNA. No ovarian metastases of endocervical adenocarcinomas were found among mucinous and endometrioid adenocarcinomas from a large chemotherapy trial of advanced stage ovarian carcinomas. The p16 staining detected in many primary ovarian adenocarcinomas in the present series seems independent from HPV oncogene activity.     

Integrated human papillomavirus types 52 and 58 are infrequently found in cervical cancer, and high viral loads predict risk of cervical cancer

OBJECTIVE: The aim of this prospective study was to analyze whether integration or high viral loads of human papillomavirus (HPV) is essential for malignant transformation of HPV types 52 and 58 as well as types 16 and 18. METHODS: Cervical swabs from 178 consecutive patients, including 81 with invasive cervical cancers and 97 with cervical intraepithelial neoplasias (CIN) II-III, were collected and examined to determine the physical status and viral load of HPV types 16, 18, 52 and 58 DNA using genechip and real-time PCR (polymerase chain reaction) analysis. RESULTS: In cervical cancer patients, the integrated form of HPV 52 and 58 DNA was found in 25.0% and 12.5% of swabs, respectively; while HPV16 and 18 DNA was found in 82.6% and 100% of swabs, respectively (P < 0.01, for pair-wise comparison of types 16, 18 versus types 52, 58).The viral loads reflected by the amount of E6 for HPV 16, 18, or 52 were significantly increased in invasive cervical cancer compared to CINII-III (P = 0.022 for type 16, P = 0.003 for type18, and P = 0.001 for type 52, respectively). Area under the receiver operating characteristic (ROC) curve for cervical cancer versus CIN II-III was 73.8%, 92.9%, and 88.5% for HPV 16, 18, and 52, respectively, indicating that real-time PCR had good diagnostic value in differentiating cervical cancer from CIN II-III. CONCLUSIONS: Infrequent integration of HPV 52 and 58 DNA in cervical cancer suggests that it is not prerequisite for progression to cervical cancer. High viral loads (E6) of HPV 16, 18, and 52 DNA may be predictive of the transition of CIN II-III to cervical cancer. Our results indicate that both viral DNA physical status and viral loads of HPV are important factors in the carcinogenesis of different HPV types.     

The relationship between c-FLIP expression and human papillomavirus E2 gene disruption in cervical carcinogenesis

OBJECTIVE: Human papillomavirus (HPV) is the essential causative factor in cervical carcinogenesis, and apoptosis inhibition is one of the key features of HPV-induced malignant transformation. This study is to investigate the possible cause-effect association between high-risk HPV and cellular FLICE-like inhibitory protein (c-FLIP), an important apoptosis regulator, during cervical carcinogenesis. METHODS: A series of 80 archival samples, including 20 squamous cervical carcinomas (SCC) 54 cervical intraepithelial neoplasia (CIN) lesions and 6 normal cervical tissues, were subjected for c-FLIP immunohistochemical staining and HPV HC-II analysis. Typing HPV-16 infection was analyzed by the polymerase chain reaction (PCR), and its status was assessed with the integrity and disruption of the HPV-16 E2 gene, which was amplified in three overlapping fragments. RESULTS: The types of HR-HPV infection and E2 disruption were associated closely with cervical lesion severity. There was a significant relationship between lesion grade and c-FLIP expression level. c-FLIP overexpression was also closely associated with HR-HPV infection and its integration status. Multivariate regression analysis revealed c-FLIP as a strong independent predictor for CIN, with 100% PPV, and showed 90.9% PPV in detecting HR-HPV, and remained a significance factor to rule out which case has no HR-HPV integration, with a 94.7% sensitivity and a 90.0% NPV. CONCLUSIONS: The present data approved that c-FLIP overexpression is related significantly to the presence of HR-HPV infection and its integration status during progression of cervical squamous cell cancer and confirmed the role of c-FLIP as an early marker of cervical carcinogenesis.     

Human papillomavirus genotyping using HPV DNA chip analysis in Korean women

This study was designed to investigate the genotypes of human papillomavirus (HPV) in Korean women who had abnormal cervical cytology and to evaluate the clinical accuracy of HPV DNA chip analysis for the diagnosis of cervical neoplasia. Liquid-based cytology preparations, HPV DNA chip analysis, and cervical biopsy were performed in 2358 women. High-risk HPV was identified in 23.5% of 1650 histologically confirmed normal samples (including cervicitis and squamous metaplasia) and in 81.8% of 708 samples with cervical intraepithelial neoplasia (CIN) and carcinoma (P<0.01). The major prevalent high-risk HPV genotypes in 381 samples of CIN II/III were HPV-16, -58, -33, and -31, in order of prevalence rate (average overall, 78.0%), and HPV-16, -18, -58, and -33 (average overall, 81.2%) in 133 samples of squamous cell carcinoma (SCC). The infection rate of HPV-16 was significantly higher than that of other high-risk HPV genotypes in all normal, CIN, and SCC cases (P < 0.01) and increased with more advanced squamous cervical lesions (P<0.01). The detection accuracy of high-risk HPV using HPV DNA chip analysis for CIN II or worse was as follows: sensitivity 84% (81-87%), specificity 72% (70-74%), positive predictive value 47% (44-50%), and negative predictive value 94% (92-95%). These results suggest that HPV DNA chip analysis may be a reliable diagnostic tool for the detection of cervical neoplasia and that there are geographic differences in the distribution of high-risk HPV genotypes.     

Clinical and prognostic significance of human papillomavirus in a Chinese population of cervical cancers

OBJECTIVE: To investigate the clinical and prognostic significance of human papillomavirus (HPV) in a Chinese population of cervical cancers. METHODS: We studied 121 cervical cancer tissue samples from patients treated at our hospital. Identification and typing of HPV were done by polymerase chain reaction (PCR) using consensus primers MY11 and MY09 followed by direct DNA sequencing. The results were correlated with various clinical and prognostic parameters. RESULTS: We found HPV DNA in 95 (78.5%) cases, including HPV-16 in 59 (48.8%) and HPV-18 in 14 (11.6%) cases. chi(2) analysis revealed no significant correlation between the presence of HPV DNA and age at diagnosis, clinical stage, histologic type, tumor grading, 2-year and 5-year survival rate. Of the factors evaluated, age at diagnosis and histologic type were found to have a statistically significant relationship with HPV type. The mean age of the HPV-18 group was 48.6 years compared to 57.1 years for the HPV-16 group (p = 0.045) and 58.2 years for the HPV-negative group (p = 0.04). HPV-18 was detected more often in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). Conversely HPV-16 was detected significantly more often in SCC (p < 0.0001). The HPV-negative group also had a higher incidence of SCC (p = 0.007). HPV-18-positive patients seemed to have more nodal involvement than both HPV-16-positive patients (45.5 vs. 20.8%) and HPV-negative patients (45.5 vs. 18.2%); however, it did not reach statistical significance. CONCLUSIONS: These observations suggest that the presence of HPV DNA does not bear any clinical or prognostic significance in a Chinese population of cervical cancers. HPV-18 is found more often in younger patients and is associated with AC.